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Cre-Lox is a powerful tool for genetic manipulation in vivo because it allows for excellent spatial and temporal control of gene expression. This is invaluable when working with animal models, where unchecked gene expression or complete gene knockout may be detrimental or lethal.

As the name suggests, the Cre-Lox system relies on two components to function: a Cre recombinase, and its recognition site, loxP. These components have been adapted from the P1 bacteriophage for use in genetic manipulation.

LoxP sites are directional 34 bp sequences made up of two 13 bp recognition sites separated by an 8 bp spacer region. The sequences don’t occur naturally in any known genomes other than the P1 bacteriophage, and are long enough that they are unlikely to occur by chance. For this reason, they can be used for specific genetic manipulations without unintended side effects.

Table 1 – Sequence of a loxP site, made up of two flanking palindromic recognition regions and a spacer region which gives it directionality.

LoxP sites are always used in pairs, often flanking a gene of interest or reporter. The orientation of the loxP sites will determine the outcome of recombination.

How does Cre-Lox recombination work? First, two Cre proteins recognize a loxP site and bind to it, forming a dimer. Two Cre-loxP dimers come together to form a tetramer, bringing the two loxP sites together with opposing directionality. Finally, dsDNA cleavage occurs in the center of the loxP site and a crossover event occurs (1). See below for a detailed diagram.

Based on the orientation of the loxP sites, there are three outcomes that can result from the recombination:

• Excision: If lox sites are in the same orientation flanking a sequence, recombination will result in the excision of the sequence, deleting it from its original locus. This process is essentially irreversible.
• Inversion: If lox sites are in opposite orientations flanking a sequence, recombination will result in the inversion of that sequence. Since the lox sites remain unchanged, this process is reversible and the gene can “flip” back and forth between both orientations indefinitely.
• Translocation: If lox sites reside on separate pieces of DNA, recombination will result in a reversible translocation event.

Lox-flanked regions of DNA are often said to be “floxed”.

The basic Cre-loxP recombination event is most useful for excision of genetic sequences, due to the irreversible nature of this event. It has been adapted for two main purposes:

Cre-dependent sequence knockout

If a sequence is flanked with two loxP sites in the same orientation, the sequence will be excised when Cre is present. This can be useful when performing gene editing experiments; successfully edited clones may be found using a selection marker, which can later be removed using Cre-Lox. However, a complete gene knockout isn’t a good choice for studying essential genes, since this would result in a lethal phenotype.

Cre-dependent gene expression

A ‘lox-stop-lox’ cassette can be placed upstream of a gene. Without Cre, the stop cassette prevents the translational expression of the gene. In the presence of Cre, the stop cassette is deleted and gene expression proceeds. However, using the ‘lox-stop-lox’ approach to control gene expression has some disadvantages. This strategy has noticeable levels of background expression when Cre is absent. As well, stop cassettes are large (~1.8 kb), which makes them difficult to package into small viruses, like adeno-associated virus (AAV) (2).

Scientists have discovered that if the spacer region of the lox site is altered, Cre doesn’t cause recombination between the wildtype and mutant lox sites. However, two of the same mutant lox site can recombine with each other. Many variations of lox site have now been developed, which are not cross-compatible:

Table 2 – Underlined nucleotides indicate those mutated from the wildtype loxP sequence. All sequences are 5’->3’.

Source: Adapted from Missirlis, et al. (3).

These sequences can be combined to make DIO (Double-floxed Inverse Orientation) or DO (Double-floxed Orientation) switches. These are also sometimes known as FLEx (or Flip Excision) switches.

DIO and DO vectors have the sequence of interest flanked by two sets of different lox sites. Two recombination steps occur – the first is an inversion of the flanked sequence using one set of lox sites. This leaves two identical sites with the same orientation on one side of the gene. The second recombination causes the excision of the intervening sequence. The order in which the lox sites undergo recombination is random, but the final product will be the same in both cases.

The inverted version of a gene will not be expressed, so DIO and DO vectors can be used to control gene expression. Unlike the large lox-stop-lox cassette, two sets of lox sites only take up ~5% of an AAV’s packaging capacity, leaving more room for other genetic elements.

A DIO vector will have the gene of interest cloned in the inverted orientation (eg. 3’-> 5’), and will become correctly oriented in the presence of Cre; thus, DIO vectors are “Cre-On”. A DO vector will have the gene of interest present in the correct orientation, and become inverted in the presence of Cre; therefore they are “Cre-Off”.

DIO and DO Switch Vectors are now available from abm! To order, please contact our technical support team.

Cre-lox recombination is ideal for performing genetic manipulation in vivo, such as in mouse models. When working in vivo, one must take into account some unique concerns:

• Breeding mice is a lengthy and laborious process.
• Mimicking a disease or mapping an organ may require a gene to be overexpressed only in certain tissues.
• Preventing lethality may require a gene to be overexpressed only after embryonic development.

Despite these difficulties, mouse models are invaluable as a research tool due to their similarity to humans. Cre-Lox recombination addresses these concerns:

• Cre-Lox is commonly used in mice, so there are many commercial Cre-expressing mouse lines available for purchase. As well, one Cre-expressing mouse line can be re-used for many different experiments, simply by changing which floxed gene is expressed from a vector.
• Tissue-specific expression in mouse models is easy to achieve with a DIO switch. A tissue-specific promoter can be used to express Cre, which will then control where the floxed gene of interest is expressed.
• Cre expression can be controlled using an inducible promoter or regulator. Cre won’t be expressed until the inducer is added, at which point the DIO/DO switch will turn on or off.

Often scientists will express either their gene of interest and/or Cre from a vector or virus, rather than from the mouse genome itself. This helps avoid lengthy breeding programs or gene editing experiments. The most commonly used virus for in vivo gene delivery is AAV because it is non-pathogenic and elicits a very mild immune response. For more information about AAV, please read our Introduction to AAV knowledge base article.

Optogenetics

A particularly popular use for DIO vectors are optogenetic experiments. In these experiments, an opsin (a neuronal channel or pump that is activated by light) can be expressed from a DIO AAV that has been injected into a mouse brain. The mouse will express Cre from a cell type-specific promoter, so that the opsin will only be expressed in certain neurons. Then, the mouse can be fitted with a fiberoptic cannula and awoken. Behavioural changes can be observed when the opsin is activated via light in those particular neurons (4).

Fate-Mapping Experiments

DIO switches can also be used for fate-mapping experiments, used to determine how certain progenitor cells will replicate as an organism grows. For these experiments, two mouse lines are crossed together: a recombinase-expressing mouse, and an indicator mouse. The recombinase mouse has Cre expressed via a promoter specific for the progenitor cell of interest. The indicator mouse will ubiquitously express a reporter such as β-galactosidase controlled by a Cre-On switch. In crossed embryos harbouring both genes, the recombinase will irreversibly turn ‘on’ expression of the indicator only in the progenitor cell of interest. All cells that originate from the progenitor cell will also express the indicator. Cre expression isn’t necessary for reporter expression in descendent cells, since the Cre-On genetic modification is irreversible and inheritable (5).

Co-Expression of Multiple Reporters

DIO vectors are also extremely useful for controlling co-expression of multiple reporters. Reporter vectors can be co-transfected at high molar ratios, while a limiting amount of Cre-expressing vector is added. This helps ensure that all cells to be labelled have been co-transfected with both reporter vectors, but that the level of expression is not too high. This is useful for mapping certain cell types, such as neurons and their synapses, which are difficult to image at high levels of reporter expression (6).

Studying Functional Mutations

Placing the lox sites in a different position can cause the activation of one gene and simultaneous excision of another. This system can be used to easily replace the wildtype version of a gene with a mutated version. This is an excellent way to study essential genes, since traditional knockout studies would be lethal.

A dual lox system can also be used to make gene knock-ins easier by using Recombinase Mediated Cassette Exchange (RMCE) (7)(8). In this approach, a selection marker would be present in the genome flanked by different lox sites. By introducing a vector carrying a gene of interest flanked by those same lox sites, the gene of interest will be swapped into the genome 50% of the time. Cells in which the swap has occurred can be enriched for or screened using the absence of the selection marker. This is a powerful tool for in vivo knock-in, since the same mouse line can be used to generate many different knock-in strains; you simply need to change the gene in your vector.

An alternate method uses inverted-repeat variants, which have been mutated in the left or right inverted repeat (called LE mutant sites or RE mutant sites, respectively) (9). Recombination will occur between a LE site and a RE site, but the result will be one wildtype loxP site and one LE/RE double mutant site. The LE/RE site is not able to undergo further recombination, so the integration is permanent.

The use of other site-specific recombination systems in combination with Cre-Lox allows for even greater flexibility and control of genetic experiments. Another popular site-specific recombination system is Flp-FRT, which uses a Flp recombinase to recombine FRT sites. However, other recombinases have also been used, including R4, lambda, phi31, HK022 and TP901-1 (1).

The use of two recombinases allows for the cloning of complex genetic switches. For example, one can target tissue-specific gene expression with even more precision by using two tissue-specific promoters – one to express Cre, and the other to express FLP. This approach can be used to express any gene of interest only at the intersection (in space or time) where Cre and FLP co-exist (10).